The present invention pertains in general to highly purified recombinant platelet-derived growth factor (rPDGF) and methods for obtaining such material. In particular, the present invention relates to affinity chromatographic purification of rPDGF employing monoclonal antibodies and to such monoclonal antibodies, as well as to a method of production of crude PDGF in quantities sufficient to be useful.
Ross et al., Proc. Natl. Acad., Sci. U.S.A. 71: 1207-1210 (1974) described platelet derived growth factor (PDGF) as a factor found in whole blood serum but not platelet-poor serum, which factor was able to support growth of fibroblasts in culture.
Unreduced PDGF is a 27-35 kd mw protein. The variation in the number of bands observed on some separating gels may be due to glycosylation differences, protease action during purification, or the presence of more than one molecular species. Reduction of PDGF yields 2 or more smaller bands on gels, in a molecular weight range of 10-18 kd. The model favored by most in the field is that the native 27-35 kd mw species consists of 2 smaller, dissimilar subunits of approximately 18 kd and 16 kd molecular weights, called respectively the "A" and "B" subunits (or alternatively PDGF A chain and PDGF B chain). Doolittle et al., Science 221: 275-76 (1983); and Waterfield et al., Nature 304: 2810-14 (1983) described the partial amino acid sequences of the two subunits of PDGF, indicating that the amino acid sequence of the PDGF B chain was more than 90% homologous with the predicted protein product of v-sis, the oncogene contained within the oncogenic simian sarcoma virus (SSV). The A chain was found to be approximately 60% homologous to the B chain.
Simian sarcoma virus was isolated from the fibrosarcoma of a woolly monkey. This virus causes oncogenic transformation of cells, and causes sarcomas in some animals. The complete SSV genome has been cloned and sequenced, and the oncogene region (v-sis) was identified and found to be potentially capable of coding for a fusion protein of 28-33 kd molecular weight. Antisera raised to peptides based on this sequence immunoprecipitated a protein of 28 kd from SSV-infected cells. This protein was called p28sis (Robbins et al., Nature 305:605-608 (1983)). Using v-sis as a probe, chromosomal clones corresponding to c-sis were isolated from a human liver library by Gallo et al. (Nature 292:31 (1981); and Josephs et al. Science 219:503-505 (1983)) and Aaronson et al. (Cell 37:123 (1983)). In addition, using v-sis as a probe, a number of human tumor cell lines were screened for expression of c-sis RNA by Gallo et al. (Nature 295:116-119 (1982)) and a high percentage of tumors of mesenchymal origin were found to contain a 4.2 kb c-sis RNA transcript.
Gallo et al. (Science 223:487-490 (1984)) disclosed the sequence of all six of the exons of the human liver c-sis chromosomal gene that are homologous to v-sis. This disclosed DNA sequence predicted a protein product almost identical to the published amino terminal sequence of the PDGF B chain. In addition, this DNA sequence predicted the remainder of the PDGF B chain amino acid sequence which had not been derived by protein sequencing.
Also, Josephs et al., Science 225: 636-639 (1984) disclosed a 2.7 kb cDNA clone from HUT102 tumor cells. While not a complete clone of the 4.2 kb RNA, it apparently contained all the sequence necessary for coding for an active PDGF B chain. When placed in a vector downstream from a SV40 early promoter, the vector was capable of transforming 3T3 cells.
PDGF and analogs thereof have been expressed in prokaryotic and eukaryotic cells transformed with vectors including exogenous genes. Murray et al., European Patent Application No. 177,957, Hannick et al., Mol. Cell. Biol., 6: 1304-1314 (1986); Hannick et al., Mol. Cell. Biol., 6: 1343-1348 (1986); King et al., Proc. Int'l Acad. Sci. (U.S.A.), 82: 5295-5299 (1985); Kelly et al., EMBO J. 4: 3399-3405 (1985); Josephs et al., Science, 225: 636 (1984); Clarke et al., Nature, 308: 464 (1984); Gazit et al., Cell 39: 89-97 (1984); and Wang, J. Biol. Chem., 259: 10645-10648 (1984). However none of these references disclose expression of more than 50 ng/ml of active PDGF in culture media. Procedures for purifying platelet PDGF include those described in Heldin et al., Nature, 319: 511 (1986); Antoniades, U.S. Pat. No. 4,479,896 and Raines et al., Methods Enzymol., 109: 749 (1985); Deuel et al., (1981). J. Biol Chem. 256: 8896-99; Antoniades, (1981). Proc. Natl. Acad. Sci. U.S.A. 78: 7314-17, however these procedures did not provide for separation of mitogenically active PDGF A homodimers or PDGF B homodimers.